1. Field of the Invention
The present invention relates generally to the fields of genetic engineering of plants using transient gene expression from viral vectors. In particular, the invention relates to the use of vectors using elements of a geminivirus to express a product of interest.
2. Description of Related Art
In recent years, there has been considerable interest in the use of transgenic plants to generate pharmaceutical proteins. A variety of compounds has been successfully expressed in plants, including viral and bacterial antigens and vaccines and as well as many different forms of antibodies (reviewed in Ma et al., 2005; Stoger et al., 2005; Ma et al., 2003). Plants are attractive as protein factories because they can produce large volumes of products efficiently and sustainably and, under certain conditions, can have significant advantages in manufacturing costs (Hood et al., 2001; Giddings, 2001). The possibility of producing therapeutic protein agents on an agricultural scale by “molecular farming” is extremely attractive. In addition to the scalability of the system, one of the major advantages of plants is that they possess an endomembrane system and secretory pathway that are similar to mammalian cells (Vitale and Pedrazzini, 2005). Thus, proteins are generally efficiently assembled with appropriate post-translational modifications. These cost and scale advantages make plant-made pharmaceuticals (PMPs) very promising for both commercial pharmaceutical production and for manufacturing products destined for the developing world.
Pharmaceutical proteins have been produced using transient, viral based expression systems (Canizares et al., 2005; Yusibov et al., 2006) or using stably transformed plants (Giddings et al., 2000; Floss et al., 2007; Twyman et al., 2005). For the latter strategy, the long time frame (months to years) to create stable transgenic plants is a concern for obtaining protein samples for initial pre-clinical studies. In addition, the lack of strong regulatory elements to drive high-level protein accumulation and the position effect associated with the randomness of transgene integration in plant genome still presents challenges for stable transgenic technology. In contrast, the transient systems that are focused on production speed have resolved the difficulty in obtaining the initial research material for testing the function of target pharmaceutical proteins in preclinical trials. For example, a “deconstructed” tobacco mosaic virus (TMV)-based three-component expression system (Marillonnet et al., 2004) allows rapid and high-yield production of vaccine candidates for subsequent immunization and challenge studies (Santi et al., 2006; Huang et al., 2006). Recently, it has been reported that full-size monoclonal antibodies (mAbs) can be rapidly produced at levels as high as 0.5 mg of mAb per gram leaf fresh weight using noncompeting viral vectors derived from TMV and potato virus X (PVX) (Giritch et al., 2006). However, the complexity of requiring five construct modules in total (two modules per subunit plus integrase) for co-expression of both heavy chain and light chain molecules (Giritch et al., 2006) may hinder the practical commercial application of this system. Furthermore, it remains a daunting challenge to find the third or more virus compatible with the existing TMV/PVX expression system to allow efficient co-expression of three or more distinct subunit proteins in same cells, which is required for the production and assembly of some important pharmaceutical complexes such as multi-component virus-like particles (VLPs) (Latham and Galarza, 2001; Pushko et al., 2005) or secretory IgA antibodies. At this point, no plant transient expression system is yet available for efficient expression of heterooligomeric proteins consisting of more than two subunits. Thus, there is a need to develop an advanced transient expression system which consists of minimum number of vectors but still permits high-level expression of multiple subunit proteins.